Lipoxygenase-catalyzed transformation of epoxy fatty acids to hydroxy-endoperoxides: A potential P450 and lipoxygenase interaction

Article abstract of DOI:10.1194/jlr.M054072

Herein, we characterize a generally applicable transformation of fatty acid epoxides by lipoxygenase (LOX) enzymes that results in the formation of a five-membered endoperoxide ring in the end product. We demonstrated this transformation using soybean LOX-1 in the metabolism of 15,16-epoxy-α-linolenic acid, and murine platelet-type 12-LOX and human 15-LOX-1 in the metabolism of 14,15-epoxyeicosatrienoic acid (14,15-EET). A detailed examination of the transformation of the two enantiomers of 15,16-epoxy-α-linolenic acid by soybean LOX-1 revealed that the expected primary product, a 13 S-hydroperoxy-15,16-epoxide, underwent a nonenzymatic transformation in buffer into a new derivative that was purifi ed by HPLC and identified by UV, LC-MS, and ¹H-NMR as a 13,15-endoperoxy-16-hydroxy-octadeca-9,11-dienoic acid. The configuration of the endoperoxide (cis or trans side chains) depended on the steric relationship of the new hydroperoxy moiety to the enantiomeric configuration of the fatty acid epoxide. The reaction mechanism involves intramolecular nucleophilic substitution (S_Ni) between the hydroperoxy (nucleophile) and epoxy group (electrophile). Equivalent transformations were documented in metabolism of the enantiomers of 14,15-EET by the two mammalian LOX enzymes, 15-LOX-1 and platelet-type 12-LOX. We conclude that this type of transformation could occur naturally with the co-occurrence of LOX and cytochrome P450 or peroxygenase enzymes, and it could also contribute to the complexity of products formed in the autoxidation reactions of polyunsaturated fatty acids.

Full text of DOI:10.1194/jlr.M054072

Lipoxygenase catalyzed transformation of epoxy fatty acids to hydroxy-
endoperoxides: A potential P450 and lipoxygenase interaction
Tarvi Tedera,b, William E. Boeglina, and Alan R. Brasha
a
Department of Pharmacology and the Vanderbilt Institute of Chemical Biology, Vanderbilt University,
Nashville, Tennessee 37232, USA
b Department of Chemistry, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia
Correspondence to Alan R. Brash, PhD, Department of Pharmacology, RRB Room 510, Vanderbilt
University Medical Center, Nashville, USA, TN 37232-6602. Tel.: 615-343-4495; Fax: 615-322-4707; E-
mail: alan.brash@vanderbilt.edu.
Running title: Lipoxygenase-epoxygenase route to hydroxy-endoperoxides
Abbreviations
AD1/AD2, 15,16-epoxy-linolenate enantiomers eluted from Chiralpak AD chiral column; EET,
epoxyeicosatrieneoic
acid;
H(P)OTrE,
hydro(pero)xyoctadecatrienoic
acid;
H(P)ETE,
hydro(pero)xyeicosatetraenoic acid; mCPBA, meta-chloroperoxybenzoic acid; LOX, lipoxygenase; RP-
HPLC, reversed phase high pressure liquid chromatography; SNi, substitution nucleophilic intramolecular;
SP-HPLC, straight phase high pressure liquid chromatography
1

ABSTRACT
Herein we characterize a generally applicable transformation of fatty acid epoxides by lipoxygenase
(LOX) enzymes that results in the formation of a 5-membered endoperoxide ring in the end product. We
demonstrated this transformation using soybean LOX-1 in the metabolism of 15,16-epoxy-α-linolenic
acid, and murine platelet-type 12-LOX and human 15-LOX-1 in the metabolism of 14,15-
epoxyeicosatrienoic acid (14,15-EET). A detailed examination of the transformation of the two
enantiomers of 15,16-epoxy-α-linolenic acid by soybean LOX-1 revealed that the expected primary
product, a 13S-hydroperoxy-15,16-epoxide, underwent a non-enzymatic transformation in buffer into a
1
new derivative that was purified by HPLC and identified by UV, LC-MS and H-NMR as a 13,15-
endoperoxy-16-hydroxy-octadeca-9,11-dienoic acid. The configuration of the endoperoxide (cis or trans
side chains) depended on the steric relationship of the new hydroperoxy moiety to the enantiomeric
configuration of the fatty acid epoxide. The reaction mechanism involves intramolecular nucleophilic
substitution (SNi) between the hydroperoxy (nucleophile) and epoxy-group (electrophile). Equivalent
transformations were documented in metabolism of the enantiomers of 14,15-EET by the two mammalian
LOX enzymes, 15-LOX-1 and platelet-type 12-LOX. We conclude that this type of transformation could
occur naturally with the co-occurrence of LOX and cytochrome P450 or peroxygenase enzymes, and it
could also contribute to the complexity of products formed in the autoxidation reactions of
polyunsaturated fatty acids.
Supplementary key words: fatty acid/physical chemistry, lipids/chemistry, NMR, fatty acid epoxide,
endoperoxide, monocyclic peroxide, epoxyeicosatrienoic acid
2

INTRODUCTION
The oxygenation reactions of polyunsaturated fatty acids include several examples in which different
enzymes collaborate in synthesis of the end product. The cyclooxygenases are paired with cytochrome
P450s in the transformation of arachidonic acid to prostaglandin endoperoxides and on to thromboxane
A2 and prostacyclin (1, 2). A P450 and cyclooxygenase participate in the opposite order in the metabolism
of P450-derived 8,9-epoxyeicosatrienoic acid (8,9-EET) by cyclooxygenase, giving epoxy-hydroxy
products (3). The plant pathway of jasmonic acid synthesis involves initial oxygenation by a 13S-
lipoxygenase (LOX) followed by transformation to an allene oxide by the P450, CYP74 (2, 4). Other
examples include the cooperation of LOX and peroxygenase enzymes in the formation of epoxy and
hydroxy fatty acids (5-7). The present work describes a new type of interaction, not so far described in
Nature, yet with the potential to produce fatty acid hydroxy-endoperoxides. Further transformations could
readily form triols or other derivatives.
The impetus for the present work arose from further studies of fatty acid epoxidation by catalase-related
enzymes primed with an oxygen donor, analogous to an earlier report of arachidonate and 8-hydroxy-
eicosatetraenoic acod (8-HETE) epoxidation by catalase-related allene oxide synthase (8). One example
involved enzymatic epoxidation of 13S-hydroxy-α-linolenic acid on the 15,16-double bond. To assign the
configuration of the new 15,16-epoxide group, we proposed a scheme using soybean lipoxygenase (LOX)
to introduce a 13S-hydroperoxide into the individual 15R,16S and 15S,16R enantiomers of 15,16-epoxy-
linolenic acid. Following reduction to the 13S-hydroxy derivatives, this would give authentic standards to
compare with the original product. While the desired primary products were recovered from the LOX-
catalyzed transformation, it was obvious that additional conjugated diene-containing derivatives were
equally abundant in the extracts. This report describes the characterization of these novel products and the
mechanism of their synthesis. Subsequent work with mammalian LOX enzymes and an arachidonic acid
epoxide (14,15-epoxyeicosatrienoic acd, 14,15-EET) demonstrate the generality of these reactions.
MATERIALS AND METHODS
Materials
α-Linolenic acid and arachidonic acid were purchased from Nu-Check Prep. mCPBA, stannous chloride,
and soybean (Glycine max) lipoxidase type V were from Sigma-Aldrich. Human 15-lipoxygenase-1 was
prepared exactly as described by expression of the recombinant enzyme in E. coli (9), and a baculovirus
construct of the murine platelet-type 12-LOX was expressed in Sf9 cells as described (10).
3

Preparation of epoxy fatty acids
Preparation of α-linolenic acid 15,16-epoxide enantiomers – α-Linolenic acid (25 mg) was epoxidized
using mCPBA, the 15,16-epoxide purified by RP-HPLC then SP-HPLC (11) and the enantiomers
resolved using a Chiralpak AD column (12) and designated as AD1 and AD2 in order of elution: these
were identified as the 15S,16R- and 15R,16S-epoxides, respectively, by CD analysis of the methyl ester
O-benzoyl derivatives as described in detail for related linoleate epoxides (13).
Preparation of 14,15-EET enantiomers – Arachidonic acid (100 mg) in 2 ml dichloromethane was
incubated with a 1.5-fold molar excess of mCPBA at room temperature for 30 min. The formed products
were taken to dryness and dissolved in MeOH prior to HPLC analysis. The arachidonate monoepoxides
are well precedented to elute in RP-HPLC in the order 14,15-, 11,12-, 8,9-, and 5,6-EET (14); 14,15-EET
was isolated by RP-HPLC using MeOH/H2O/HAc 85:15:0.01 (v/v/v) on a Waters Symmetry C18 column
(4.6 x 250 mm; 5 µ). The enantiomers of 14,15-EET were resolved using a Chiralcel OJ column (4.6 x
250 mm; 5 µ) and a solvent of Hexane/MeOH/HAc (100:0.5:0.1, v/v/v, run at 2 mL/min), eluting in the
order 14S,15R as peak 1 and 14R,15S as peak 2 as reported (15).
Incubations with soybean LOX-1
The individual enantiomers of 15,16-epoxy-linolenate were reacted at room temperature with soybean
LOX-1 (76 nM) in 0.5 mL 0.1 M K2HPO4 (pH 8.7) buffer in a quartz cuvette and the change in the
absorbance recorded by repetitive scanning (200 – 300 nm) using a Lambda-35 UV/Vis Spectrometer
(Perkin-Elmer). Addition of enzyme led to the appearance of a conjugated diene chromophore (ε ~ 25,000
M-1 cm-1) over the course of 10 min, with a faster reaction rate for the AD2 enantiomer (14R,15S-epoxy).
To reduce hydroperoxide products in situ, incubations were conducted in the presence of 0.5 mM SnCl2.
All the formed products were acidified with 1M KH2PO4 and 1N HCl down to pH 5 and extracted on 1 cc
HLB Oasis cartridges and eluted with MeOH prior to HPLC analyses. To isolate sufficient products for
NMR analyses, the incubations were scaled up to 20 ml 0.1 M K2HPO4 buffer containing 152 nM
soybean LOX-1. The methyl ester derivatives of isolated products were prepared using diazomethane.
Incubations with platelet 12-LOX and 15-LOX-1
The individual enantiomers of 14,15-EET were incubated with platelet 12-LOX (0.42 µM) in 0.5 ml 50
mM Tris pH 7.5 buffer containing 150 mM NaCl and the changes in the absorbance recorded by
repetitive scanning (200 – 300 nm) using a Lambda-35 UV/Vis Spectrometer (Perkin-Elmer). Addition of
enzyme led to the appearance of a conjugated diene chromophore over the course of 5 min (ε ~ 25,000),
with similar reaction rates for both enantiomers of 14,15-EET. To reduce hydroperoxide products in situ,
incubations with platelet 12-LOX were conducted in the presence of 0.5 mM SnCl2. All the formed
products were extracted on 1 cc HLB Oasis cartridges as previously described. Subsequent incubations
4

with human 15-LOX-1 were carried out in a similar manner. The methyl esters of products formed by
platelet 12-LOX and human 15-LOX-1 were prepared using diazomethane. For the NMR analyses, the
incubations with platelet 12-LOX were scaled up to 10 ml buffer and use of 0.4 µM enzyme. The methyl
ester derivatives of the human 15-LOX-1 products were reacted with 1.5 equivalents of TPP in MeOH for
30 min at room temperature for 10 min prior to HPLC and GC-MS analyses.
HPLC analyses
The formed products were conducted on SP-HPLC (Hex/IPA/HAc, 100:3:0.1, by volume for free acids
and Hex/IPA, 100:2 for methyl esters) using a Thomson silica column (4.6 x 250 mm, 5µ). A flow rate of
1 ml/min was used and UV signals at 205, 220, 235, 270 nm recorded using a Agilent 1100 series diode
1
array detector. To prepare sufficient products for H-NMR, about 0.5 mg of fatty acids were injected for
the collection. Each collected product was dissolved in MeOH and kept at -20°C until further analysis.
LC- and GC-MS analysis
LC-MS analysis of soybean LOX-1 catalyzed products - The products formed from the enantiomers of
15,16-epox-linolenate on incubation with soybean LOX-1 and LOX-1/SnCl2 were eluted on RP-HPLC
(CH3CN/H2O/HAc, 45:55:0.01, by volume; 0.4 mL/min) using a Kinetex C18 column (3 x 100 mm, 2.6µ)
and molecular weights were established from the M-H anions measured by negative ion electrospray
LTQ1 ion trap LC-MS instrument.
GC-MS analysis of human 15-LOX-1 catalyzed products – The methyl esters of hydroperoxy-EETs were
treated with TPP to prepare the corresponding hydroxy-EETs and repurified by SP-HPLC. The methyl
esters of hydroxy-EETs dissolved in 200 µL ethyl acetate were hydrogenated using H2 with ~1 mg 5% Pd
on alumina for 3 minutes at room temperature. The catalyst was removed by extraction with water and
centrifugation and the organic phase containing the saturated hydroxy-C20 fatty acids was transfered for
further derivatization. GC-MS analyses of the methyl ester trimethylsilyl derivatives of the reduced and
hydrogenated hydroperoxy-EETs was carried out in the positive ion electron impact mode (70 eV) using a
ThermoFinnigan DSQ mass spectrometer. The initial temperature was set for 180°C and then increased to
300°C at 20°C/min and then held at 300°C for 4 min.
NMR analysis
1
1H NMR and H,1H COSY NMR spectra were recorded on a Bruker 600-MHz spectrometer at 298 K.
The parts/million values are reported relative to residual non-deuterated benzene (C6D6; δ = 7.15 ppm).
All spectra were analyzed on Bruker TopSpin 3.0 software. In addition to NMR analyses, 3D models of
the formed products were created using ACD-Labs software (Supplementary data).
5

RESULTS
Reaction with soybean LOX-1 with the 15,16-epoxides of α-linolenic acid
The 15,16-epoxide enantiomers of α-linolenic acid were prepared as outlined in Methods and for
convenience here are designated as AD1 (15S,16R) and AD2 (15R,16S), in order of their elution from the
Chiralpak AD column. The 15,16-epoxides retain a 9cis,12cis pentadiene in the structure and are potential
substrates for soybean LOX-1. Figures 1A and 1B illustrate transformation of the two mirror image
15,16-epoxides with the LOX-1 enzyme as observed by UV spectroscopy. Both enantiomers give rise to a
typical conjugated diene chromophore, with a much slower reaction occurring with AD1, the 15S,16R
isomer, (cf. Fig. 2A, 2B); under the conditions used here, AD1 has an initial turnover of 2 s-1 and AD2 of
13 s-1, in the order of 1% and 5% of the rates with α-linolenic acid itself (16).
Subsequent SP-HPLC analyses revealed a more complex pattern of products than initially expected, with
three products recovered from a 10 min reaction of AD1 (Fig. 2A, top chromatogram), and two products
from AD2 (Fig. 2B, top). All these products showed UV absorbance at 235 nm and contained a
conjugated diene chromophore (Supplementary Fig. S1). The product pattern was time-dependent, such
that upon 20 min incubation in the reaction buffer with soybean LOX-1, there was an increase in the
relative abundance of the earliest-eluting product (Fig. 2AB, lower chromatograms, products 1 and 4).
To help understand the basis for the appearance of multiple products containing a conjugated diene
chromophore, the incubations with soybean LOX-1 were repeated in the presence of stannous chloride
(SnCl2) that will reduce hydroperoxides to the hydroxy derivative in situ. This simplified the product
pattern such that only two conjugated dienes appeared from AD1 and only one product from AD2 (Fig.
2C). It appeared that the SnCl2-catalyzed reduction of the hydroperoxy products of the soybean LOX-1
reaction prevented the time-dependent transformation that gives rise to the earliest-eluting products (1 and
4 in Fig. 2A and 2B).
Structural analysis of soybean LOX-1 products 1 – 8
LC-MS analyses (negative ion mode, Supplementary Fig. S2) showed that all of the products formed in
the initial incubations with soybean LOX-1 (products 1 – 5) have a M-1 ion at m/z 325, corresponding to
the addition of molecular oxygen to the 15,16-epoxy-linolenate. All the SnCl2-reduced products each
have a M-1 ion at m/z 309, corresponding to retention of one of the oxygens from O2.
Fragmentation of the peroxide products provided the first indications of the presence of an endoperoxide
moiety in the structures of products 1 and 4. Whereas the other peroxide products showed only a
prominent M-1 ion at m/z 325 in the negative ion mass spectrum, 1 and 4 contained an equally prominent
6

ion at m/z 237, interpreted as loss of C15-C18 (88 a.m.u.) and compatible with the presence of an oxygen
attached to C15 in addition to the oxygen at C16.
All the products described so far were prepared in sufficient amounts for NMR analysis, which revealed
their covalent structures, and based on literature precedents, the relative stereochemistry of the carbon
chain substituents of the endoperoxides in products 1 and 4 (17, 18) (Table 1). The NMR spectra and
COSY analyses are given in Supplementary data, pages 10 - 18. Scheme 1 shows the structures of the
peroxide products 1 – 5 and the deduced pathway to the endoperoxides (products 1 and 4). The AD1
enantiomer (15S,16R-epoxy) reacts relatively slowly with soybean LOX-1 and is converted to three
products: one is the 13S-hydroperoxide (product 3), which is expected, and this is further transformed to
the endoperoxide (1); in addition, the slowly-reacting AD1 enantiomer also gives the 9-hydroperoxide (2)
as an additional product. The faster reacting AD2 enantiomer only gives the 13S-hydroperoxide (5) and
the corresponding endoperoxide (4). The SnCl2 reduced products are the corresponding 13-hydroxy-
15,16-epoxy derivatives (7 and 8), plus the 9-hydroxy-15,16-epoxide (6) formed from AD1.
Non-enzymatic formation of the endoperoxides
The 13-hydroperoxy-15,16-epoxides (2 and 5) were stable in organic solvents and could be purified with
conventional HPLC methods at room temperature. Re-incubation of the purified hydroperoxy-epoxides in
the original pH 8.5 buffer in the absence of enzyme led to a perceptible change in the UV profile (Fig.
3A); this is attributed to their non-enzymatic transformation to the corresponding endoperoxides, which
have a higher lambda max (239 nm, versus 237 nm of the hydroperoxy derivatives) (Fig. 3B). This
transformation in buffer alone matches the time-dependent changes noted earlier upon extending the 10
min incubations to 20 min (Fig. 2AB, upper and lower chromatograms). Formation of the endoperoxides
in the absence of enzyme was confirmed by extraction and HPLC-UV analysis of the products (data not
shown).
There is a noticeable difference in the rate of endoperoxide formation from the 13S-hydroperoxy
derivatives of the linolenic acid AD1 and AD2 15,16-epoxides. Whereas AD1 reacts slower with soybean
LOX-1 (Fig. 1A, 1B), its product (13S-hydroperoxy-15S,16R-epoxy) shows the faster non-enzymic
transformation to the endoperoxide. This is understandable in that the newly formed hydroperoxy moiety
is better positioned for SNi attack on the 15,16-epoxide (as explained in Discussion).
Testing the generality of the endoperoxide formation
To test the generality of the endoperoxide formation from fatty acid hydroperoxy-epoxides we used two
mammalian LOX enzymes and the enantiomers of 14,15-EET as substrate. Reactions of the platelet-type
12-LOX with 14,15-EET are analogous to the linolenic acid transformations described above. On the
7

other hand, the primary oxygenation activity of 15-LOX-1 is not possible using 14,15-EET as substrate;
in this case it is the minor 12 dioxygenase activity of 15-LOX-1 that comes into play, as well recognized
before in the 15-LOX-1-catalyzed transformations of 15S-HPETE to leukotriene epoxides (19) and the
related transformations catalyzed by the leukocyte-type 12-LOX (20).
Reactions with mouse platelet-type 12-LOX
Reaction of recombinant platelet-type 12-LOX with the enantiomers of 14,15-EET was observed by
repetitive UV scanning, which showed the appearance of a conjugated diene chromophore (cf. Fig. 1A,
1B) and rates similar for the two enantiomers and comparable to the rate of reaction of arachidonic acid.
The turnover values (kcat) for 14S,15R-EET and 14R,15S-EET were 1.3 s-1 and 0.95 s-1, respectively, and
1.8 s-1 for arachidonic acid. SP-HPLC analysis of the products revealed the formation of two major
products from each 14,15-EET enantiomer, Fig. 4A, 4B. As found before with the α-linolenate epoxide
products, there is a time-dependent transformation of the more polar 10 and 13 to the earlier-eluting
products 9 and 12. NMR analysis (Supplementary data, pages 14 - 17) of all four main compounds
confirmed what would be predicted, namely that the more polar 10 and 13 are 12-hydroperoxy-14,15-
epoxides, and the time-dependent secondary products 9 and 12 are 12,14-endoperoxy-15-hydroxy
derivatives from 14S,15R-EET and 14R,15S-EET, respectively. Whereas the two 14,15-EET isomers
react similarly with the 12-LOX, the secondary transformation to the endoperoxides is faster with the
14S,15R enantiomer (cf. upper and lower chromatograms of Fig. 4A and 4B), compatible with its more
favorable stereochemistry for the SNi attack of the hydroperoxide on the epoxide carbon at C14 (see
Discussion).
The Supplementary data also illustrates that the UV spectra of the four conjugated dienes show distinct
differences with a higher lambda-max and smoother profile for the earlier eluting endoperoxide
derivatives (Supplementary Fig. S3A), and that in the presence of SnCl2 only a single product, the
corresponding hydroxy-epoxide (11 and 14) is formed from each 14,15-EET enantiomer (Supplementary
Fig. S4).
Reactions with human 15-LOX-1
Using arachidonic acid as substrate, 15-LOX-1 displays a number of reactions and secondary
transformations. The primary dioxygenase activities give 15S-HPETE and 12S-HPETE in ~10:1
proportions (21) and secondary reactions give 5,15- and 8,15-diHPETEs as well as 14,15-LTA4 and its
hydrolysis products (19). Understanding these properties help in explaining a slightly more complex
profile of products formed from the enantiomers of 14,15-EET. Lipoxygenase catalysis of 14,15-EET to
the 12-hydroperoxide requires an initial hydrogen abstraction from C10, which is a secondary property of
15-LOX-1, giving rise normally to the minor proportion of 12-HPETE from arachidonate. Therefore it
8

was not unexpected that the two 14,15-EET enantiomers are transformed by recombinant human 15-
LOX-1, albeit at a rate about an order of magnitude slower than using arachidonate as substrate. The
products were separated by SP-HPLC and the structures determined by GC-MS of the methyl ester TMS
derivative after reduction and hydrogenation of the double bonds (Supplementary Fig. S5).
The 14S,15R enantiomer is the slightly faster reacting (1.0 s-1 versus 0.3 s-1 for 14R,15S-EET, and 10 s-1
for arachidonic acid), and it forms three main products (Fig 4C). The tallest peak (10) is the 12-
hydroperoxy-14,15-epoxide and it shows a time-dependent transformation to the earlier-eluting
compound 9, which was shown by UV analysis and HPLC mobility to be identical to the 12S,14R-
endoperoxy-15R-hydroxy product of 12-LOX (compound 9). The “extra” peak in Fig. 4C, compound 15,
was identified by GC-MS (after reduction of the hydroperoxide) as an 8-hydroxy-14,15-epoxide (Scheme
2, upper half).
The second 14,15-EET enantiomer (14R,15S) was converted by 15-LOX-1 to 13, identical to the 12S,14S-
endoperoxy-15S-hydroxy product of 12-LOX, and which showed very little further transformation to an
endoperoxide (12, a very small peak in Supplementary Fig. S6, lower chromatogram). The major peak
(16) was identified by GC-MS (after reduction) as a 5-hydroxy-14,15-epoxide (Scheme 2, lower half).
9

DISCUSSION
Routes to hydroxy-endoperoxides - Fatty acid epoxides are typical products of cytochrome P450
enzymes (22-24) and we describe a generally applicable reaction in which the fatty acid epoxide is further
oxygenated by a LOX enzyme followed by the non-enzymatic formation of a mono-cyclic endoperoxide
(Scheme 3). In principle the order of the first two reactions could be reversed, with initial formation of the
hydroperoxide and secondary epoxidation (Scheme 3, lower half). Peroxygenase enzymes are an
alternative source of the fatty acid epoxide: typical plant peroxygenases use a fatty acid hydroperoxide to
“charge” the enzyme and effect intermolecular oxygen transfer and epoxidation (5-7, 25). This co-
occurrence of the LOX and epoxygenase activities together presents favorable circumstances for the
further transformation to hydroxy-endoperoxides. It is interesting to consider that the cyclooxygenase-
catalyzed oxygenation of 8,9-epoxyeicosatrienoic acid referred to in the Introduction sets up the potential
for the synthesis of an 8-hydroxy-9,11-endoperoxide synthesis – however the concomitant peroxidase
activity of PGH synthase rapidly reduces the initial 11-hydroperoxy moiety and the product recovered is
the 8,9-epoxy-11-hydroxy derivative (3).
Mechanism of formation of the endoperoxides - The step of endoperoxide synthesis is understandable
as an intramolecular nucleophilic substitution (SNi-type) between the hydroperoxy nucleophile and the
epoxy electrophile (Scheme 4). Differences in the rate of endoperoxide formation depend on the relative
configuration of the hydroperoxy group to the epoxide moiety. The rate is faster when the hydroperoxide
is more favorably position for SNi attack on the epoxide (Scheme 4, left side). For example, although the
15S,16R-epoxy isomer of α-linolenate (AD1) reacts slower with soybean LOX-1 (Fig. 1A, 1B), the
oxygenation product (13S-hydroperoxy-15S,16R-epoxy) shows the faster non-enzymic transformation to
the endoperoxide (Scheme 4, left side). The same observations applied to the step of endoperoxide
synthesis involving the 12-hydroperoxy-14,15-epoxy products of 12-LOX and 15-LOX-1.
There is a precedent for this fatty acid endoperoxide synthesis in the chemical literature. Porter and
coworkers used mCPBA and a catalytic amount of trichloroacetic acid in methylene chloride to convert 9-
hydroperoxy-octadeca-6,10,12-trienoic acid (9-HPOTrE-ω6) to multiple products including a 6-hydroxy-
7,9-endoperoxide (26). The mechanism clearly involved the intermediacy of the direct mCPBA-catalyzed
formation of the 6,7-epoxide of the 9-hydroperoxide which was converted by the trace of acid to the
corresponding hydroxy-endoperoxide. Earlier, Corey had suggested a related theoretical pathway of
prostaglandin H2 synthesis is which an arachidonate 14,15-epoxide could be oxygenated and cyclized to
an endoperoxide with final opening of the epoxide ring to give the 9,11-endoperoxy-15-hydroxy product
(27). Another related transformation involves formation of 5-hydroxy-prostaglandin I1 from 5,6-EET via
COX-1 catalysis: synthesis of the corresponding endoperoxide and its reduction to a PGF analog permits
α
2
10

nucleophilic attack of the C9 hydroxyl on the 6-carbon of the 5,6-epoxide, giving the 5-hydroxy-PGI1
isomers as final products (28).
The reactions we characterize take place in an aqueous environment and being enzyme-catalyzed, the
products have a specific stereochemistry. Even though the endoperoxide formation is non-enzymatic, the
reaction is stereoselective – a chiral epoxide and hydroperoxide giving a single endoperoxide
diastereomer via intramolecular nucleophilic substitution. The reaction occurs most readily when the
epoxide oxygen is on the opposite face of the carbon chain to the attacking hydroperoxide, allowing ready
SN substitution and producing an endoperoxide with side-chains in the cis configuration (Scheme 4, left
side). When the epoxide oxygen is on the same face as the hydroperoxide, the reaction is slower and the
product is a trans endoperoxide (Scheme 4, right side). Although the transformation is slightly favoured
in more alkaline solution which we detected using incubation buffers (pH 7.5 vs 8.5) for corresponding
soybean LOX-1 and mammalian LOXs, the water- and time-dependent interaction is primarily
determined by the stereochemistries of the epoxy and hydroperoxy moieties.
Comparison and contrast with autoxidative formation of endoperoxides - It is well recognized that
non-enzymatic peroxidation can give rise to hydroperoxy-monocyclic endoperoxides (29, 30). Reduction
of such hydroperoxy-endoperoxides will give a racemic version of the hydroxy-endoperoxide products we
describe. In addition, several authors have used the initial LOX-catalyzed formation of 13-hydroperoxy-
linolenic acid to study its further non-enzymatic transformation to hydroperoxy-endoperoxides (31, 32).
The direct formation of hydroxy-endoperoxides is also theoretically feasible via lipid peroxidation
because autoxidative reactions can include the production of fatty acid epoxides (33, 34), which are then
open to a non-enzymatic peroxidation to the hydroxy endoperoxides as we have characterized.
Trihydroxy fatty acids and other predicted derivatives – The fatty acid monocyclic endoperoxides are
relatively stable under mild conditions (e.g. HPLC at room temperature). Also, unlike hydroperoxides, the
monocyclic endoperoxide moiety is resistant to reduction using mild reducing agents such as
triphenylphosphine and even sodium borohydride (31). Nonetheless, in biological systems opening of the
epoxide ring is likely to occur on exposure to peroxidases, in which case the products will be fatty acid
triols. The same triols could arise also from hydrolysis of the precursor hydro(pero)xy-epoxides, which
are likely substrates for epoxide hydrolase (35).
Epoxy fatty acids as LOX substrates – A side issue of some interest in relation to our results is the
suitability of specific fatty acid epoxides as substrates for LOX catalysis. One of the central concepts of
substrate binding in lipoxygenases is the head-to-tail orientation of the fatty acid, which in the case of the
soybean LOX-1, human 15-LOX-1 and the platelet-type 12-LOX involves a “tail-first” entry into the
11

LOX active site (36). Our results indicate that the presence of an epoxide on the tail carbons slows the
rate of reaction but does not interfere with the specificity of the oxygenation. As the reaction rate of the
platelet-type 12-LOX with 14,15-EET is comparable to the rate with arachidonic acid, this raises the
possibility of their co-occurrence and interaction in the cardiovascular system.
Conclusion
The dioxygenation of epoxy fatty acids by lipoxygenases give rise to hydroperoxy- and hydroxy-epoxy
derivatives. Subsequent transformation of hydroperoxy-epoxides to hydroxy-endoperoxides as a result of
intramolecular nucleophilic substitutions between hydroperoxy and epoxy moieties is an alternative way
of synthesizing monocyclic peroxides in vitro. In vivo, the crosstalk between epoxygenase and
lipoxygenase pathways might result in hydroperoxy-epoxy and corresponding endoperoxy derivatives that
might undergo to reduction, giving variety of hydroxy fatty acids. The presence and biological
significance of described fatty acid derivatives might be a matter of future research in lipidomics.
_____________________________________________
Acknowledgements
We thank Prof. Nigulas Samel for encouragement and support for these studies and Prof. Ned Porter for
providing helpful mechanistic insights. We also thank Dr. Wade Calcutt and Brian Hachey for assistance
with the mass spectrometric analyses. This work was supported by NIH grants GM-15431 and GM-
74888, by a voucher for use of the mass spectrometry resource through the Vanderbilt-Ingram Cancer
Center Support grant (P30 CA068485), and by the Institutional Research Grant IUT19-9 from the
Estonian Ministry of Education and Research
Supplemental Information (Separate file)
12

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14

Table 1. 1H-NMR chemical shifts (δ) and coupling constants (J) of cis- and trans-endoperoxides formed
in the soybean LOX-1 (left side) and platelet-type 12S-LOX (right side) catalyzed metabolism with
15,16-epoxy-α-linolenic acid and 14,15-EET, respectively. Characteristically different values of cis and
trans are shown in bold.
Soybean LOX-1/15,16-epoxide
Platelet-type 12S-LOX/14,15-EET
δ (ppm)
cis / trans
H13
J (Hz)
δ (ppm)
cis / trans
H12
J (Hz)
cis / trans
cis / trans
J13,12 = 7.6 / 7.4
J13,14a = 7.5 / 7.0
J13,14b = 7.6 / 6.8
J14a,13 = 7.5 / 7.4
J14a,15 = 7.7 / 4.6
J14a,14b = 12 / 11.9
J14b,13 = 8 / 5.9
J14b,15 = 5.2 / 8.3
J14a,14b = 12 / 11.9
J15,14a = 7.9 / 4.7
J15,14b = 5.5 / 8.3
J15,16 = 5.6 / 4.7
J12,11 = 7.7 / 7.4
J12,13a = 7.6 / 5.5
J12,13b = 7.6 / 5.5
J13a,12 = 7.6 / 7.6
J13a,14 = 7.6 / 4.8
J13a,13b = 11.9 / 12
J13b,12 = 8 / 5.2
J13b,14 = 5.2 / 8.1
J13a,13b = 12.1 / 11.9
J14,13a = 7.9 / 5.2
J14,13b = 5.3 / 8.1
J14,15 = 5.3 / 5.2
4.56 / 4.64
4.58 / 4.59
H14a
H13a
2.17 / 2.37
2.24 / 2.27
H14b
H13b
1.87 / 1.97
1.91 / 1.98
H15
H14
3.88 / 3.94
3.91 / 3.96
15

Figure legends
Figure 1. Repetitive UV scans of the formation of products from the α-linolenate 15,16-epoxides AD1
(A) and AD2 (B) by soybean LOX-1. Turnover numbers are presented in the text.
Figure 2. SP-HPLC analyses of products (analyzed as methyl esters) from 15,16-epoxides AD1 (A) and
AD2 (B) by soybean LOX-1, and (C) from identical incubations with soybean LOX-1 carried out in the
presence of SnCl2. The top chromatogram in A is from a 10 min incubation with enzyme and the lower
chromatogram from a 20 min incubation. In B, the incubation times were 3 min and 10 min. In panel (C),
the hydroxy products 6, 7, and 8 are analogues of hydroperoxy derivatives 2, 3, and 4, respectively.
Samples were run on a Thomson 5µ silica column (250 x 4.6 mm) with a solvent of hexane/IPA (100:2
by volume) at a flow rate of 1 ml/min with UV detection at 235 nm. The structures of products 1 – 5 are
shown in Scheme 1.
Figure 3. The non-enzymic formation of endoperoxides from 15,16-epoxide AD2 (A). Formation of
endoperoxides from other hydroperoxy-epoxides can be detected in a similar manner due to the
characteristic UV spectra of endoperoxides (B).
Figure 4. SP-HPLC analyses for products (analyzed as methyl esters) from incubation of platelet 12-LOX
with 14S,15R- (A) and 14R,15S-EETs (B) after 10 and 30 min, and (C) the products from incubation of
human 15-LOX-1 with 14S,15R-EET. (Incubation of 15-LOX-1 with 14R,15S-EET is given in the
Supplement, Fig. S5). Chromatographic conditions were as in Fig. 2. The structures of the products are
shown in Scheme 2.
Scheme 1. Products formed from α-linoleate 15,16-epoxides AD1 and AD2 by soybean LOX-1.
Scheme 2. Products formed from 14,15-EETs by platelet 12-LOX and human 15-LOX-1.
Scheme 3. The potential interaction between epoxygenase and lipoxygenase pathways.
Scheme 4. The reaction mechanism for the formation of endoperoxides from epoxy fatty acids.
16

Figure 1
AD1 substrate
AD2 substrate
A
B
O
O
1.2
1.0
0.8
15 16
15 16
1.0
4 & 5 min
3 min
11 min
9 min
7 min
2 min
0.8
0.6
5 min
1 min
AU
AU
0.6
0.4
0.2
0.0
3 min
0.4
0.2
0.0
2 min
1 min
15 sec
15 sec
280
300
200
220
240
260
320
200
240
260
280
300
320
220
Wavelength (nm)
Wavelength (nm)

Figure 2
endoperoxide
1
A
10 min
mAU
80
2
9-OOH
60
40
20
AD1
soybean LOX
13-OOH
3
0
20 min
175
125
75
Formation of
endoperoxides
(faster)
25
0
235 nm
0
2
4
6
8
10
min
13-OOH
5
mAU
160
B
3 min
120
80
AD2
soybean LOX
endoperoxide
4
40
0
10 min
160
120
80
40
0
Formation of
endoperoxides
(slower)
235 nm
0
2
4
6
8
10
min
C
mAU
30
13-OH
7
AD1
soybean LOX
with SnCl2
9-OH
6
20
10
0
13-OH
8
AD2
soybean LOX
with SnCl2
40
20
0
235 nm
0
4
6
8
2
10
min

Figure 3
A
AU
0.65
B
AU
0.16
OOH
O
H
monocyclic peroxides
(smoother)
0.55
0.45
0.35
0.25
0.12
0.08
0.04
0
30 sec
O
O
OH
typical conjugated
diene chromophore
(13S-OOH-15,16-epoxide)
H
8 min
0.15
0.05
200
210
230
240
250
260
270
260 270 280 290 nm
220
nm
220 230 240 250

Figure 4
A
10 min
endoperoxide
9
mAU
500
12-OOH
10
14S,15R-EET
+ 12-LOX
300
100
12-OH
11
0
600
30 min
400
200
Formation of
endoperoxides
(faster)
235 nm
0
8
0
2
6
min
4
12-OOH
13
B
10 min
mAU
600
14R,15S-EET
+ 12-LOX
400
200
endoperoxide
12
12-OH
14
0
600
400
200
30 min
Formation of
endoperoxides
(slower)
235 nm
0
0
min
2
4
6
8
C
mAU
125
12-OOH
10
14S,15R-EET
+ 15-LOX-1
10 min
100
75
50
25
0
75
50
25
0
12-OOH
endoperoxide
8-OOH
15
9
20 min
Formation of
endoperoxides
235 nm
0
1
2
3
4
5
6
7
8
9

Scheme 2
OOH
O
platelet 12S-LOX
& human 15-LOX-1
O
14 15
12
14 15
Initial reaction
14S,15R-EET
12S-OOH-14S,15R-EET (product 10)
Secondary reaction
(faster)
human 15-LOX-1
O
O
OH
HOO
O
12 14
14 15
8
15
H
H
15R-OH-12S,14R-endoperoxide (product 9)
8S-OOH-14S,15R-EET (product 15)
platelet 12S-LOX
& human 15-LOX-1
OOH
O
O
14 15
14 15
12
Initial reaction
14R,15S-EET
12S-OOH-14R,15S-EET (product 13)
human 15-LOX-1
Secondary reaction
(slower)
O
O
OH
HOO
O
12 14
14 15
5
15
H
H
5S-OOH-14R,15S-EET (product 16)
15S-OH-12S,14S-endoperoxide (product 12)

Scheme 1
OOH
O
O
soybean LOX-1
13
15 16
15 16
Initial reaction
(slower)
13S-OOH-15S,16R-epoxide (product 3)
AD1 (15S,16R-epoxide)
Secondary reaction
(faster)
soybean LOX-1
HOO
O O
13 15
OH
O
16
H
15 16
9
H
9S-OOH-15S,16R-epoxide (product 2)
16R-OH-13S,15R-endoperoxide (product 1)
OOH
O
O
15 16
soybean LOX-1
13
15 16
Initial reaction
(faster)
AD2 (15R,16S-epoxide)
13S-OOH-15R,16S-epoxide (product 4)
Secondary reaction
(slower)
O
O
OH
13 15
16
H
H
16S-OH-13S,15S-endoperoxide (product 5)

Scheme 3
PUFA
P450 or
Peroxygenase
O
LOX
(or autoxidation)
OOH
O O OH
O
P450 or
Peroxygenase
OOH
LOX
(or autoxidation)
PUFA

Scheme 4
O
O
R
S
S
R
H H
H H
LIPOXYGENASE
OOH
OOH
O
O
Intramolecular nucleophilic
substitution
(SNi-type reaction)
OO
OO
O
O
(faster)
(slower)
O O
O O
OH
OH
H
H
H
H